Breeding of leafy vegetables like lettuce and spinach aims at the production of commercial varieties optimally adapted to local growing conditions which allows the grower to maximise the productivity of high quality produce. Many characteristics need to be taken into account during selection which relate to both input as well as output traits. One of the most important input traits in this respect relates to disease resistance, in particular to resistance towards oomycetes and more in particular towards downy mildews.
The outcome of the interaction of a plant with a pathogen depends on many genetic factors both of the pathogen as well as the plant. In order to infect a plant successfully, a pathogen needs to overcome a number of barriers.
The first layer is of a physical nature and can be manifested in the form of an enforced cell wall or cuticle layer.
As a second layer of defense, a plant can exhibit a basal form of resistance which may prevent the pathogen from infecting the plant. Non-host resistance can be considered as an extremely successful form of basal defense which in fact is effective for most plant pathogen interactions.
In case the first two barriers have been overcome by the pathogen, a third layer of intricate defense may be encountered in the form of the induction of factors which actively inhibit the infection process initiated by the pathogen. In many different plant pathogen interaction systems such as the interaction of lettuce or spinach with downy mildews, the plant initiates these events only after specific recognition of the invading pathogen. In many cases this recognition occurs after the pathogen has established the first phases of interaction and transferred a so called pathogenicity (or avirulence) factor into the plant cell.
These pathogenicity factors interact with host components in order to establish conditions which are favorable for the pathogen to invade the host and thereby cause disease. When a plant is able to recognize the events triggered by the pathogenicity factors a resistance response can be initiated.
Recognition of these events occurs directly or indirectly by resistance gene (R-gene) products produced by the invaded plant for which recently a mechanistical model, the so-called guard model, has been proposed (Dangl J. L. and Jones, J. D. G. (2001) Nature 411, 826-833). Upon recognition a multicomponent cascade of events takes place including the generation of reactive oxygen species (ROS) leading to a tightly regulated local induction of programmed cell death around the cells which have been infected by the pathogen.
Additionally, genes encoding defense factors such as pathogenesis related or PR proteins are induced which contribute to the execution of the defense response. Also increased callose formation can be induced by recognition of pathogen attack.
Furthermore, the localisation of a pathogen at sites of attempted invasion leads to a systemic induction of the defense response which is called systemic acquired resistance or SAR.
Co-evolution of the plant and the pathogen has led to an arms race in which the resistance can be broken down as a consequence of the capability of the pathogen to interact with and modify alternative host targets or the same targets in a different way. In any case, the recognition is lost and infection can be established successfully resulting in disease. In order to re-establish resistance in a plant, a novel resistance gene has to be introduced which is able to recognize the mode of action of an alternative pathogenicity factor.
Traditionally, plant breeders have been very successful in generating downy mildew resistant lettuce and spinach varieties by making use of resistance genes residing in the wild germ plasm of the crop species.
As the resistance evoked by the R-genes is highly effective, R-genes are exploited at large scale in commercial plant breeding. As a consequence of their mode of action these resistances are not durable as the pathogen population constantly adapts to the newly introduced R-gene. For lettuce, this has resulted in the introduction of over 20 different R-genes in commercial varieties over the last 50 years. As resistance towards downy mildew is a prerequisite for any cultivar to be commercialised, resistance breeding has been given high priority.
As the commercial value of a particular lettuce or spinach variety is primarily determined by its resistance towards the prevailing downy mildew pathotypes in the growing area, the development of novel varieties is largely determined by the ability and velocity of a plant breeder to introgress appropriate downy mildew resistances into the commercial varieties. Furthermore, as the occurrence of novel resistance breaking strains is largely unpredictable, the commercial value of a variety can either last long or diminish rapidly.
Commercial success in lettuce or spinach breeding is therefore largely determined by the availability of effective resistance genes i.e. those genes able to prevent infection by the prevailing downy mildew pathotype, as well as the efficiency of resistance breeding. Thus, a large effort in lettuce and spinach breeding is dedicated towards downy mildew resistance which is primarily beneficial to the crop grower and which may go at the expense of quality traits beneficial to the consumer of fresh produce.
Due to the low durability of the R-gene mediated resistance, a large proportion of the breeding resources in lettuce and spinach has to be allocated towards breeding for downy mildew resistance. It is therefore clear that there exists a need in the art to have available sources of downy mildew resistance in lettuce and spinach which are much more durable as compared to the R-gene mediated resistance. Moreover, it is desirable to have more alleles available that can add to the resistance of a plant against oomycetes.
In the research that led to the present invention, the inventors contemplated that in order to achieve a more durable form of resistance towards downy mildew in lettuce and spinach, other mechanisms than those based on the R-gene mediated recognition and subsequent response should be exploited. As mentioned, several layers of defense exist in a plant which need to be broken down by a pathogen in order to establish disease. More durable forms of resistance may therefore be achieved which act independently of each other and of the specific interaction of an R-gene product and the pathogenicity factor host complex.
For example, it has been shown to be feasible to modify a plant which displays a constitutive form of defense. This means that the defense system is switched on irrespective of inductive signals coming form a successful recognition of a pathogen by a host R-gene product. By modifying factors controlling this response, constitutive activation can be achieved. This can be done through downregulation of repressors or by ectopic activation of inducers of the resistance response. Several methods are available to the person skilled in the art to achieve such downregulation of repressors or ectopic activation of inducers.
In many known cases, however, as a consequence of the constitutive activation of the defense response, resources are reallocated towards defense factors which leads to a significant reduction of plant growth. In commercial crop breeding this yield penalty is obviously not acceptable. Furthermore, part of the defense response can be manifested in the form of the synthesis and accumulation of secondary metabolites which may be lowering the nutritional value of produce or may even be harmful to the health of the consumer.